Cdc34 protein of Saccharomyces cerevisiae, a ubiquitin conjugating enzyme, can undergo autoubiquitination. The reaction product is a multiubiquitinated Cdc34 protein. Attachment of a multiubiquitin chain to a protein is usually a signal for selective degradation of that protein. Whether this is the case for Cdc34 protein is a subject of the proposed research. We propose to identify the autoubiquitination sites in Cdc34 protein and then alter these sites by in vitro mutagenesis to generate autoubiquitination-defective mutants. We will then express these mutants in Saccharomyces cerevisiae to determine whether autoubiquitination serves to target Cdc34 protein for degradation and whether such degradation is important in controlling the availability of Cdc34 protein during the cell cycle. Finally, we will further define the biochemical role of CDC34 by identifying and characterizing proteins that interact with Cdc34 protein, either as participants in a multiprotein complex or as substrates for Cdc34-dependent ubiquitination. These studies will lead to important insights into the role of ubiquitination in the control of cell growth.